a) Field of the Invention
The invention enables spatial fixation of microscopic objects in laser scanning microscopes, also during the displacement of the object plane, for example, when recording an image. Therefore, moving objects can also be imaged sharply.
b) Related Background
Optical tweezers have proven to be an important work tool for a range of biological work techniques. Expanded experimental possibilities can be anticipated due to the combination of laser scanning microscopes with laser micro-techniques.
LSM recordings of moving objects, above all in the interior of unopened cells, often do not result in satisfactory images because many subcellular structures move during the scanning time. Ideally, the optical tweezers can be used for careful (vital) fixation. Further, a spatially defined displacement of fixed objects is possible with the optical tweezers. Application examples for the use of compensated optical tweezers in the laser scanning microscope are the examination of organelles, for example, chloroplasts, or the determination of objects which are moved by motor proteins. In the latter case, it is even possible under suitable conditions to take energy measurements. In principle, moving objects, for example, particles in suspension or determined organelles, can not be imaged sharply without being fixated by compensated optical tweezers.
Optical tweezers which are coupled in through the objective have their focus in the object plane. When there is a parallel displacement of the object plane due to the three-dimensional image recording process (scanning), the focus of the optical tweezers is also displaced. As a result, objects that are held by the optical tweezers are likewise displaced. However, this is undesirable during image recording. Therefore, the displacement of the object plane must be compensated by a suitable device in the beam path of the optical tweezers.
One aspect of the present invention is to fulfill a need in laser scanning microscopes of different manufacturers whenever the optical tweezers are coupled in through the objective and the third dimension is made accessible during image recording by the displacement of the objective or object stage or by another method which displaces the focus of the optical tweezers relative to the specimen.
When optical tweezers are coupled into an inverted microscope via a second high-aperture objective which couples in the optical tweezers from the other side of the specimen (K. Visscher, G. J. Brakenhoff, “Single Beam Optical Trapping Integrated in a Confocal Microscope for Biological Applications”, Cytometry 12:486-491)), a compensated movement of the optical tweezers is rendered superfluous. However, the specimen must be located between two glass coverslips for this purpose and may not exceed a certain thickness. Further, this type of in-coupling limits conventional microscope applications because the objective for the optical tweezers is placed at the location of the transmitted light beam path in the inverted microscope. Moreover, there is no longer unlimited free access to the specimen from above, which makes applications with microinjection devices or temperature regulating devices, for example, very difficult, if not impossible. This is also true for constructions in which optical fixation of particles is carried out by glass fibers which are provided with microlenses and which are guided directly onto the specimen. In addition, problems arise with respect to the sterility of the specimen because the glass fibers must be immersed in thicker liquid layers when particles are to be fixated on the underside of the liquid. An aspect of the invention is to address these problems as well.